Hematopoiesis is organized as a cellular hierarchy initiated by rare self-renewing hematopoietic stem cells (HSC), which in turn produce lineage-committed progenitors and eventually all the mature blood cells. It is important to identify the molecular events and pathways regulating hematopoietic stem and progenitor self-renewal, growth, death, and differentiation. However, it has become increasingly clear that studying cell populations only provides average values, and that interrogation of individual cells could reveal the presence of subpopulations and provide insight into these biological processes that are masked at the population level. The main goals for this grant application are to combine the power of microfluidic genomic and epigenomic analysis with the ability to isolate purified hematopoietic stem and progenitor populations to develop a high resolution understanding of hematopoiesis. This proposal aims to use single cell microfluidic PCR to determine the heterogeneity of known populations, and to functionally characterize new intermediate populations. Next, this proposal aims to investigate hematopoiesis in situ to define and molecularly characterize hematopoietic niches. Finally, this proposal aims to use the gene signatures of defined mouse progenitors to identify human homologues. The surface proteins expressed within each cell type will be used to separate human cells by FACS, and transplantation into NSG mice will indicate their function. Deep molecular analyses of these mouse and human progenitors to identify candidate gene loci that regulate critical biological functions. The analyses will include definition of open versus closed chromatin-associated genome sequences, definition of the methylome for each progenitor and comparison with the RNA expression profiles, and development of new bioinformatic tools to aid in the identification of critical molecular factors. Functional assays will be conducted with siRNA to investigate the most interesting genes, and will also include microfluidic nanoculture to assess the effect of soluble factors on progenitor functions. Finally, within the NHLBI Progenitor Consortium human bone marrow disorders will be examined from the standpoint of an expanded knowledge of human hematopoietic progenitors.